Immune-stimulatory mAbs are currently being evaluated as antitumor agents. Although overall toxicity from these agents appears to be moderate, liver toxicities have been reported and are not completely understood. We studied the effect of systemic CD40 antibody treatment on myeloid cells in the spleen and liver. Naïve and tumor-bearing mice were treated systemically with agonistic anti-CD40 antibody. Immune cell subsets in the liver and spleen, serum transaminases, and liver histologies were analyzed after antibody administration. Nox2^−/−, Cd40^−/−, and bone marrow chimeric mice were used to study the mechanism by which agonistic anti-CD40 mediates its effects in vivo. Suppressor function of murine and human tumor-induced myeloid-derived suppressor cells (MDSC) was studied upon CD40 ligation. Agonistic CD40 antibody caused liver damage within 24 hours after injection in two unrelated tumor models and mice strains. Using bone marrow chimeras, we demonstrate that CD40 antibody–induced hepatitis in tumor-bearing mice was dependent on the presence of CD40-expressing hematopoietic cells. Agonistic CD40 ligation–dependent liver damage was induced by the generation of reactive oxygen species. Furthermore, agonistic CD40 antibody resulted in increased CD80-positive and CD40-positive liver CD11b⁺Gr-1⁺ immature myeloid cells. CD40 ligation on tumor-induced murine and human CD14⁺HLA-DR^low peripheral blood mononuclear cells from patients with cancer reduced their immune suppressor function. Collectively, agonistic CD40 antibody treatment activated tumor-induced myeloid cells, caused myeloid-dependent hepatotoxicity, and ameliorated the suppressor function of murine and human MDSC. Collectively, our data suggest that CD40 may mature immunosuppressive myeloid cells and thereby cause liver damage in mice with an accumulation of tumor-induced hepatic MDSC. Cancer Immunol Res; 3(5); 557–66. ©2015 AACR.